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Q: Fachverband Quantenoptik und Photonik

Q 41: Ultrashort Laser Pulses: Applications III

Q 41.6: Vortrag

Mittwoch, 10. März 2010, 17:45–18:00, F 342

STED nanoscopy of living hippocampal neurons in organotypic brain slices — •Nicolai T. Urban¹, Katrin Willig¹, U. Valentin Nägerl², and Stefan W. Hell¹ — ¹Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany — ²INSERM U862/Université Victor Segalen Bordeaux 2, Bordeaux, France

Stimulated Emission Depletion (STED) nanoscopy is a light microscopic technique that enables fluorescence imaging with nanoscale resolution. In this approach the diffraction barrier is overcome by overlapping the excitation with a doughnut-shaped, red-shifted STED beam, thereby removing the fluorescence ability of the fluorophore in the outer region of the excitation spot, leaving only a nanosized region in which the fluorophore is able to signal.

However, when attempting to image neurons deep inside the sample, spherical aberrations stemming from the refractive index mismatch between the brain tissue and the widely used oil immersion severely limit the penetration depth. The use of glycerol (n=1.45) as an immersion liquid instead lessens the refractive index mismatch noticeably. The remaining aberrations can be compensated down to a certain depth by a correction collar on the objective.

Using this approach we recorded YFP-labeled actin filaments in dendritic spines, which are protrusions on the dendrites, of living hippocampal neurons in organotypic brain slices. Spine rearrangement was imaged over long time periods in depths down to 50 µm with nanoscale resolution of < 80 nm.